Direct observation of DNA overwinding by reverse gyrase

Taisaku Ogawa, Katsunori Yogo, Shou Furuike, Kazuo Sutoh, Akihiko Kikuchi, Kazuhiko Kinosita

    Research output: Contribution to journalArticle

    15 Citations (Scopus)

    Abstract

    Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70°C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71°C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50°C. The unloaded reaction rate at 71°C exceeds five turns per second, which is >10<sup>2</sup>-fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s<sup>-1</sup>, indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼5pN·nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.

    Original languageEnglish
    Pages (from-to)7495-7500
    Number of pages6
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume112
    Issue number24
    DOIs
    Publication statusPublished - 2015 Jun 16

    Keywords

    • DNA overwinding
    • Magnetic tweezers
    • Reverse gyrase
    • Topoisomerase
    • Torsion

    ASJC Scopus subject areas

    • General

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